| Hepatitis C virus (HCV) is a member of the Flaviviridae family, with a positive sense, single-stranded RNA genome of ~ 9.6 kb. The HCV genome consists of 5'-untranslated region(5'-UTR), a single open reading frame(ORF) containing 10 genes, and 3'-untranslated region(3'-UTR). The ORF codes for a polyprotein of 3010~3030 amino acid (aa) residues, which is cleaved by cellular and viral proteases to produce at least 10 mature viral protein products, including core(C), envelope 1(E1), E2, p7, nonstructural protein(NS2), NS3, NS4A, NS4B, NS5A, and NS5B. Recently, an alternative open reading frame(ARF) was found, overlapping the HCV core protein-coding sequence. The ARF codes for a novel HCV protein, named F protein. The F protein localizes predominantly to the endoplasmic reticulum(ER ) membranes, with a half-life of 5~10 minutes,and its function is unclear.In this study, three aspects of work were performed, such as the expression of F protein in prokaryocyte, the association of F protein with cell apoptosis, and the effect of phosphorylation on the distribution of F protein in cells. We aim to search for the relationship between F protein and HCV pathogenicity ,and provid theoretical evidence for prevention from and management of HCV infection.1. Prokaryotic expression, purification, and antigenicity of HCV F proteinFirstly, according to the codon appetite in Escherichia coli, eleven pairs of overlapping primers was designed and used to synthesize the full-length HCV f gene , from which the truncated HCV f65 gene fragment was amplified by PCR. HCV f65 gene was then cloned into pET32a(+), and transformed into E.coli strain Plyss(DE3). The above recombinant E.coli were induced by IPTG for the production of HCV F65 protein, with a molecular weight of 32 kilo-dalton (kDa). The HCV F65 protein, mainly expressed in the bacteria in soluble form. After the induction by 0.6mM and 1mM IPTG, the soluble F65 protein occupied 33.4% and 34.2% of the total protein in bacteria cell. The expressed HCV F65 protein, purified by Ni-NTA agarose, was found no other protein band by SDS-PAGE, and its concentration was from thirty-eight to seventy-two micro-gram per millilitre (μg/ml). In western blotting test, the purified F65 protein reacted with anti-histidine (His) antibody, and not with anti-green fluorescence protein(GFP) antibody, indicating that the F65 protein was specific in antigenicity. Secondly, the purified F65 protein, with a concentration of 50μg/mL, was used as capture antigen in ELISA to detect anti-F antibody in serum from health people, HBV-infected patients, and HCV-infected patients(n=30). The result showed that, in the serum from the health people, HBV-infected patients, and HCV-infected patients (n=30), the mean A450 value of anti-F antibody were 0.044±0.011, 0.054±0.023, and 0.125±0.061, with significant difference in HBV-infected and HCV-infected patients(p﹤0.001). The cut-off value for anti-F antibody equal to two and one-tenth times of the mean A450 value of negative control group, health people(n=30) as negative control group, and the calculated cut-off value was 0.092. According to the resultant cut-off value(0.092), the positive rate of serum anti-F in HCV-infected and HBV-infected patients were 63.3%(19/30) and 3.3%(1/30), respectively, significantly higher serum anti-F prevalence in HCV-infected patients than in HBV-infected patients(p﹤0.001). Finally, the expressed HCV F65 protein was further used to immunize rabbit for making of polyclonal anti-F. The rabbit polyclonal anti-F was purified by Staphylococcus aureus protein A agarose. The rabbit-derived polyclonal anti-F specifically reacted with HCV F65 protein, with the title of 1:30000, but not with thioredoxin(Trx) protein from pET32a(+) vector. This part of work indicates that (1)Our expressed HCV F65 protein is of antigenicity, and can be used to determine serum anti-F; (2)Anti-F IgG does exist in sera from HCV-infected patients; (3)Moreover, the rabbit-derived polyclonal anti-F can be used to detect HCV F protein.2. Proliferation and anti-apoptosis induced by tumor necrosis factor-α(TNF-α)in HepG2 cells stably transfected with HCV f geneAfter the recombinant pcDNA3.1+-f plasmids expressing HCV F protein were transfected into HepG2 cells by liposome transfection, the HepG2 cells stably expressing HCV F protein were produced under the management of G418(350μg/ml), named Hep-f cells. In the same way, the HepG2 cells stably transfection with empty pcDNA3.1+ vector were produced, named Hep-3.1 cells, and used as control. The HCV F protein, with a molecular weight of 14kDa was confirmed to exist in the first generation Hep-f cells by WB, as well as ten generations cells. Secondly, the growth of Hep-f and Hep-3.1 cells were examined by MTT method, and the growth rate of Hep-3.1 cells was significant lower than of Hep-f cells(p﹤0.01), reflecting the fact that the HCV f gene promotes multiplication of HepG2 cell. Moreover, after treatment with 20,40,60,100,and 200 international units per millilitre (IU/ml) TNF-α, the growth of Hep-f and Hep-3.1 cells were investigated by MTT, and the inhibition rates of growth were 4.8%~19.4% and 8.3%~53.1%, with the former lower than the latter(p﹤0.05), suggesting that Hep-f cells are resistant to the effect of TNF-α.Treatment of the Hep-f and Hep-3.1 cells with 100 IU/ml TNF-αfor 18 or 36 hours(h), then the apoptosis cells were double labelled by Annexinⅴ/PI and analyzed by flow cytometry, every test independent performed for five times , and the apoptosis rate equaled to mean apoptosis rate plus/ minus standard deviation(x±s). When 100 IU/ml TNF-αtreatment for 18h, the apoptosis rate of Hep-f and Hep-3.1 cells were ( 0.41±0.11 ) % and ( 37.43±2.03 ) %, respectively, with significant difference(p<0.001). When 100 IU/ml TNF-αtreatment for 36h, the apoptosis rate of Hep-f and Hep-3.1 cells were (10.03±0.41)% and (44.63±3.37)%, respectively (p<0.001).Moreover, Hep-f and Hep-3.1 cells treated with 100 IU/ml TNF-αfor 18h, then marked with a fluorescence dye of Hochest33342, its nucleus were observed by laser confocal microscope. The results showed that abnormal nucleus forms emerged in Hep-f and Hep-3.1 cells, such as pyknosis, and formation of apoptotic body, but the percent of abnormal nucleus forms occurring in Hep-3.1 cells was higher than the percent in Hep-f cells. The above two kinds of results reflect that Hep-f cell generates resistance to the apoptosis induced by TNF-α.In order to look for the relationship between the apoptosis resistance phenotype of Hep-f cell and the activation of nuclear factor-κB(NF-κB), treatment the Hep-f and Hep-3.1 cells with 100 IU/ml TNF-α, then the amount of p65 (a subunit of NF-κB) and IκB-αprotein were determined by WB at 0h,0.5h,1h,2h,18h, and 36h. The results showed that, at 0h,0.5h,1h,2h,18h, and 36h, (1) the amount of p65 in whole Hep-f cells were 2.85,2.67,2.61,2.84,2.80 and 2.69, and that were 2.88, 2.94, 2.86, 2.76, 2.71 and 2.50 in Hep-3.1 cells, with no significant difference(p>0.05); (2) the amount of p65 in Hep-f cell nucleus were 1.10,3.90,3.92,3.92,2.07 and 1.67, and that were 1.08,2.05,2.04,1.95,1.03 and 1.10 in Hep-3.1 cell nucleus, with significant difference (p<0.05). Moreover, the curve of p65 content in Hep-f cell nucleus contained three phases, rising sharply to 3~4 times of the level before treatment (0~0.5h), retaining the peak level (0.5~2h), and decreasing slowly but above the level before treatment(2~36h). With regard to Hep-3.1 cells, the curve of p65 content in nucleus contained four phases, rising sharply to 1~2 times of the level before treatment (0~0.5h), retaining the peak level (0.5~2h), descending slowly to the level before treatment(2~18h), and retaining the level before treatment (18~36h); (3) the amount of IκB-αin Hep-f cell cytoplasm were 0.46,0.37,0.19,0.25,0.32 and 0.34, and that were 0.45,0.39,0.31,0.39,0.5 and 0.52 in Hep-3.1 cell cytoplasm, with significant difference(p<0.05). In Hep-f cell, the IκB-αcontent decreased in the period of 0~1h, and rised slowly but under the level before treatment in the period of 1~36h. In Hep-3.1 cell, the IκB-αcontent decreased in the period of 0~1h, then rised slowly in the period of 1~2h but near to the level before treatment at 2h, and retained the level before treatment in the period of 2~36h. Our conclusion is that the apoptosis resistance phenotype of Hep-f cell attributes to the abnormal activation of NF-κB by HCV F protein.3. Association of intercellular distribution of F Protein with its mutation in phosphorylation sitesOn the basis of the phosphorylation sites of HCV F protein predicted by NetPhos2.0 Server software, overlapping primers were designed and used to synthesize the HCV mutated f (mf)gene, in which the codons 13,14,114,121 and 124 sites were mutated from TCT to GAT. Consequently, the amino acids shifted from serine(S) to aspartate(D). The recombinant pEGFP-mf and pEGFP-f were constructed by cloning the mf gene and f gene into pEGFP-N1, respectively. The resultant recombinant plasmids were transfected into HepG2 cells by liposome transfection, and the distribution of mutant-type F protein or wild-type F protein in HepG2 cells was examined by laser confocal microscope. The results showed that: (1) In HepG2 cells, the mutated F protein was mainly located in nucleus(90%), only 10% of which in cytoplasm(10%); the mean fluorescence intensity of mutated F protein in nucleus and cytoplasm were 63.70±3.20 and 7.06±0.34, respectively, with significant difference(p<0.001, t =99.2). (2) On the contrary, the wild-type F protein was mainly located in cytoplasm(94.9%), partly in nucleus(5.1%); the mean fluorescence intensity were 83.34±4.07 and 4.48±0.22, respectively(p<0.001, t =106.5). From the above results, we conclude that: (1) In cells, there are phosphorylation and non-phosphorylation forms of HCV F protein, which are different in location. (2) The phosphorylated F protein lies mainly in cytoplasm, involving in the regulation of HCV replication, and the non-phosphorylated F protein mainly in nucleus, with an effect on cell genetic transcription.
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